This invention relates to a process for producing N-acyl-D-phenylalanine esters.
Heretofore, the resolution of racemic D,L-phenylalanine mixtures has followed three different routes of optical resolution in order to obtain one of the optically active antipodes. The first includes asymmetric hydrolysis of N-chloroacetyl-D,L-phenylalanine by the carboxypeptidase in pancrease. A second involves asymmetric hydrolysis of N-acetyl-D,L-phenylalanine by mould aminoacylase which process can be operated in a continuous fashion using a packed column. And, finally, a physico-chemical resolution based on preferential crystallization of isomers from supersaturated solution of acetyl-D,L-phenylalanine ammonium salt. In addition, there are several synthetic methods for production of L-phenylalanine. For example, conversion of L-tyrosine, treatment of synthetic phenylpyruvic acid with transaminase and enzymatic isomerization of synthetic D,L-phenylalanine by microorganisms. Kaneko et al, Synthetic Production and Utilization of Amino Acids, J. Wiley & Sons, pp. 171-179 (1974).
In Belgian Patent 855,051, there is taught a method and a composition for treatment of D,L-amino acids to resolve the racemic mixture and obtain L-isomer in which a supported aminoacylase is used to treat aqueous solutions of the D,L-amino acid. The support is a porous inorganic substrate of specified grain size, surface area, pore diameter and volume and is coated with a network polymer film, or carries a tertiary amine or quaternary ammonium salt group. Typical supports are titania, alumina or silica. The network polymer can be polyamino epoxides, polyamineformaldehyde, phenol-formaldehyde mixtures and polymerized mixtures of vinyl monomers. The aminoacylases are enzymes of animal orgin, such a pork kidney extracts or microorganism products, such as from Aspergillus, Lactobacillus arabinosus, Micrococcus glutamicus, and Pseudomonoas cruciviae. The process includes contacting the amino acids, such as N-acetyl-D,L-amino acid, with the complex of support/tertiary amine/polymer network/enzyme, for example, by passing through a packed column.
In U.S. Pat. No. 3,963,573, there is taught a process for producing optically pure N-acyl-L-methionine by subjecting an N-acyl-D,L-methionine ester to the action of a proteolytic enzyme selected from the group consisting of sulfhydryl proteinases and microbially derived serine proteinases and separating the resulting N-acyl-L-methionine. The art has recognized that certain proteolytic enzymes can be produced in a pure foam, such as from Bacillus subtilis, Guntelberg Trav. Lab. Carlsberg, Ser. Chim. Vol. 29, p. 36 (1954). The proteolytic enzyme prepared from the strain of Bacillus subtilis was purified by crystallization and its physico-chemical properties were determined. The enzymatic properties were investigated insofar as optimum pH for milk coagulation, stability, degradation of casein, hydrolysis of hemoglobin, activators and inhibitors for the enzymes, the effect on ovalbumin and other characteristics. In the Journal of Biological Chemistry, Vol. 243, No. 7, pp. 1344-1348 (1968), Barel, examined the activity of Carlsberg and Novo subtilisins toward a number of N-acetylamino acid esters and amino acid esters. The enzymes were also compared with respect to their efficiency in catalyzing aminolysis reactions, their rates of inactivation by certain aromatic sulfonyl halides and the rates of deacylation of their N-trans-cinnamoyl derivatives. Although the enzymes were found qualitatively indistinguishable from the standpoint of substrate specifity, significant quantitative differences were observed. Thus, the microbially derived serine proteinases, for example, Novo and Carlsberg subtilisin exhibited varying of degrees of esterase activity on various N-acyl-L-amino acid esters. However, there is not disclosed a process for resolution of N-acyl-D,L-phenylalanine esters employing the activity of serine proteinases.
D-phenylalanine heretofore had little, if any, use. However, it has recently been found that D-phenylalanine is a simple non-addictive analgesic compound that stimulates the body's own pain-fighting system, provides significant relief in cronic-pain patients and produced long term analgesia. Therefore, a process for producing N-acyl-D-phenylalanise esters and, particularly, N-C.sub.1-9 acyl-D-phenylalanine esters which can be converted easily to D-phenylalanine would be very desirable. An especially effective process would involve (1) readily available and inexpensive material, such as enzyme, and (2) produce high purity material in high yield at a rapid rate.